All organisms must accurately replicate their genomes to faithfully pass their genetic information onto subsequent generations. Within the cell, however, DNA is crowded by a large number of DNA-binding proteins that can act as obstacles to DNA synthesis. The collisions that occur when the DNA replication machinery collides with other proteins, including transcribing RNA polymerase, are known to stall the replication fork and result in recombination events that are a hallmark of cancer. Despite previous biochemical studies that have explored the problems of replicating through crowded DNA substrates, little is known about the mechanistic details of collisions between the replication and transcription machineries. An immediate goal of this proposal is to directly observe the outcomes of collisions between a model viral DNA polymerase and singly-bound protein roadblocks, such as RNA polymerase, lac repressor, and catalytically inactive EcoRI, which have all been demonstrated to impede the replication fork in vivo. These studies will rely on single molecule optical microscopy and DNA curtain technology, developed in the Greene lab, to directly visualize collisions between the replication machinery and protein obstacles. Another aspect of this proposal is the development of a single molecule assay by which collision experiments can be performed with tandem arrays of bound proteins as is more reflective of a crowded physiological setting. The results from these studies with both singly-bound and tandemly-arrayed protein obstacles will ultimately lead to collision experiments with the more complicated multicomponent replisomes from E. coli and S. cerevisae, which will provide further details of collision- induced replication fork stalling and recombination in higher organisms. This work will help reveal the mechanisms by which the replication machinery deals with protein obstacles, and offers the potential to reveal aspects of these collisions that cannot be more easily accessed through other methods.